Method for instrument determination of a measured variable which changes with time

ABSTRACT

The present invention relates to a method for instrument determination of measured variable L(t) which changes with time, the intention being to determine the maximum rate value of L(t) in a region with a linear reaction profile, and the time as well as the duration of the reaction time window being variable with the linear region and depending on the nature of the reaction and the reaction conditions. Specifically, the present invention relates to the determination of protein concentrations with the aid of light scatter, which is produced by specific antibodies, in homogeneous solutions. In particular, the invention relates to reactions which take place slowly and have a largely linear profile over a relatively long time, the rate of formation of antigen-antibody complexes in the linear section of the reaction (V MaxLin ) being determined as the measured variable.

This is a continuation of application Ser. No. 08/936,544, filed Sep.24, 1997 now U.S. Pat. No. 6,044,330.

BACKGROUND OF THE INVENTION

The present invention relates to a method for instrument determinationof a measured variable L(t) which changes with time, the intention beingto determine the maximum value of L(t) in a region with a linearreaction profile, and the time as well as the size of the reaction timewindow being variable with the linear region and depending on the natureof the reaction and the reaction conditions. Specifically, the presentinvention relates to the determination of protein concentrations withthe aid of light scatter, which is produced by specific antibodies, inhomogeneous solutions. In particular, the invention relates to reactionswhich take place slowly and have a largely linear profile over arelatively long time, the rate of formation of antigen-antibodycomplexes in the linear section of the reaction being determined as themeasured variable.

The phenomenon of light scatter on particles in a homogeneous medium isused for concentration determination, both by measuring the scatteredlight intensity (nephelometry) and by measuring the intensity loss ofthe light beam passing through the medium (turbidimetry).

The immunochemical reaction between a soluble antigen and a bivalent orpolyvalent antibody leads to large groups of molecules which scatterlight to a major extent. The time profile of such reactions veryfrequently corresponds to the general kinetic profile of successivefirst order reactions and has a point of inflection, so that the maximumrate of reaction does not occur until during the course of the reaction(see, for example, FIGS. 1a-1 c). A concentration-dependent measurementsignal can be obtained in various ways from the signal-time curves inFIGS. 1a-1 c.

The intensity of the signal change can be increased by bonding one ofthe reaction partners to particles, for example in the“particle-enhanced assays” known per se to a person skilled in the art.

In the end-point method, the measurement signal is determined at a timewhich is so late that, on the basis of experience, it is no longerchanging but no precipitation is taking place. In the “fixed time”method, the actual measurement method is the difference between twosignals which are determined at times that are different but are fixedin advance.

In the kinetic “peak rate method”, the maximum rate of reaction(V_(Max)), that is to say the maximum change (δ) of the signal (S) perunit time (δt), is determined

a) by measurements of δS at sufficiently short time intervals (δt) anddetermination of the maximum quotient δS/δt,

b) electronic differentiation δS/δt and determination of the maximum,

c) construction of the tangent to the signal/time curve anddetermination of the maximum gradient S=signal, t=time.

The method according to the invention can in principle be used for alldeterminations of a measured variable which varies with time, providedthe change in the measured variable is linear only in a sub-region andis intended to be used for evaluation of the linear part.

A large number of analytes can now be quantified by direct or indirectscattered light measurement using the described methods. If oneconsiders the dependency of a suitable measurement signal on theconcentration of a reaction partner, for example the antigen, while theother reaction partner is used with a constant concentration, then, forexample, it is possible in the case of immunochemical reactions toobserve that the same measurement signal can be caused by both a lowconcentration and a high concentration of the analyte. This leads to anambiguity in the signal concentration relationship, which is known tothe person skilled in the art as the antigen excess phenomenon“high-dose hook” or Heidelberger curve. This ambiguity can in principlebe observed wherever complexes of different stoichiometry are possible,depending on the excess amount of one reaction partner or the other, andthe signal characteristic, for example scattered light, of thesecomplexes does not differ.

Such immunochemical determination methods are known per se to the personskilled in the art, for example from EP 0 252 127.

In addition to the possible ambiguity in the signal concentrationrelationship, a further problem is the determination of lowconcentrations and the evaluation of reactions which take place slowly.The reaction profile of the antigen-antibody bonding in principle has alag-phase at the start, a region where the rate of reaction is a maximumand a saturation region (FIGS. 1a-1 c). The extent to which these threephases are pronounced is very heavily dependent on the concentration ofthe antigen of the antibody and, furthermore, on a large number of otherfactors, such as the temperature and the dilution medium, although theseare kept as constant as possible in a test system.

The present invention was thus based on the technical problem ofproviding an immunochemical determination method with the aid of lightscatter produced by specific antibodies, which method not only allowsvery low concentrations to be measured but also offers a high level ofprotection against the “high-dose hook” effect.

This technical problem is solved by the provision of the embodimentsdescribed in the claims.

The essential part of the method according to the invention is that themeasurement time window of the respective reaction is adapted bysuitable technical steps such that the evaluation takes place reliablyin the linear region and the region of the maximum rate of reaction ofthe time-dependent reaction. The result of such evaluation is V_(MaxLin)which sometimes is also called X_(lin).

The method according to the invention can be ensured by varioustechnical embodiments.

Analytes for the purposes of the invention are plasma proteins such asFerritin, PSA, IgA, IgG and proteins which can be ascribed to the fieldof coagulation, such as D-Dimer and clotting factors, in particulargenetic variants of clotting factors and, furthermore, haptenes such ashormone and messenger peptide.

The method according to the invention can also advantageously be usedfor determining the functionality of the clotting system, such as quicktest and aPTT; that is, an activated partial thromboplastin time.

It has thus surprisingly been found that the determination methoddescribed in the following text not only allows the measurement of lowconcentrations but also ensures increased protection against the“high-dose hook” effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a(1) to 1 c(3) show concentration dependent measurement signalsof Ferritin v. time. The various concentration of ferritin (in μ/l) areas follows: FIG. 1a(1)-137.6; FIG. 1a(2)-17.2; FIG. 1a(3)-2.15; FIG.1b(1)-68.8; FIG. 1b(2)-8.6; FIG. 1b(3)-1.08; FIG. 1c(1)-34.4; FIG.1c(2)-4.3; and FIG. 1c(3)-0.54.

FIG. 2 shows the main run reaction window as a function of the initialrun V_(Max).

FIGS. 3a to 3 d show the V_(MaxLin) method with a two-stage evaluationusing several samples. FIGS. 3a and 3 c show low-concentration sampleruns. FIGS. 3b and 3 d show normal-concentration sample runs.

FIG. 4 shows a determination of the precision (CV, %), the deviationfrom the nominal value (% deviation), and the linearity (correlationcoefficient) versus nominal area.

FIGS. 5a to 5 d show kinetics approximated using third order polynomialand its first derivative. FIGS. 5a and 5 b show one low-concentrationrun and its first derivative. FIGS. 5c and 5 d show onehigh-concentration sample run and its first derivative.

FIG. 6 shows a comparison of signal precision.

FIG. 7 shows intra assay variation coefficient at low analateconcentration.

FIG. 8 shows a reference curve.

FIGS. 9a and 9 b show the linearity of a dilution series.

FIG. 10 shows a comparison of V_(MaxLin) wit a fixed time evaluation forseveral samples.

FIG. 11 shows antigen excess.

FIG. 12 shows the application of V_(MaxLin) on different assay types.

FIGS. 13a and 13 b show results of a Von Willebrand-Assay.

The method according to the invention is carried out in two stages, asample being measured and the stored reaction kinetic being used foreach stage. In the first stage, a relatively short time window is usedto determine the initial maximum rate of reaction. The length of thetime window t_(Test) is preferably set such that, on average and over aplurality of measurements and batches, it corresponds to the length, ofthe linear reaction section for the reaction kinetics of samples whoseconcentration is at the upper end of the measurement range. It alsobecomes clear from this that the size of the initial reaction window isa test-specific parameter which is dependent, inter alia, on the analyteas well as the test system or verification system used. The V_(MaxPre)determined in this way is used to determine the optimum reaction timewindow. The dependency between V_(MaxPre) and the optimum reaction timewindow t_(Lin) must be determined in a test-specific manner inpreliminary experiments (FIG. 2). The important factor in this case isthat a reaction window is determined for each V_(MaxPre), that on theone hand includes as many measurement points as possible, but on theother hand also considers only points which are on the linear section ofthe reaction.

Various methods can be used to determine the test-specific dependency.The important factor in this case is that the dependency betweenV_(MaxPre) and the reaction time window size is defined such that theprecision and correctness are optimized. This dependency can bede-fined, for example, by measurements on a pool of serum samples ofdifferent analyte concentration or serum standards with differentdilution. The concentrations in this case advantageously extend over theentire desired measurement range. In a first step, all the samples aremeasured, the kinetics are stored and the values of V_(MaxPre) aredetermined. Different correlation coefficients are now obtained for eachsample measurement by changing the reaction window step by step. Thewindow size having the best correlation coefficient is that which alsotakes best account of the linear section of the reaction. Anothersuitable measure_of linearity is the average distance between themeasurement points and the linear regression line. Thus, first of all,the best t_(Lin) for linearity is obtained for each sample with aspecific V_(MaxPre). The result for all samples with differentV_(MaxPre) is a relationship between V_(MaxPre) and the main runreaction window size t_(Lin) for the best linearity in the reactionwindow. By way of example, FIG. 2 shows the relationship between themain run reaction window size t_(Lin) and V_(MaxPre) for the bestcorrelation co-efficient.

This relationship is used to produce a reaction curve with standardserum dilution steps of known concentration (V_(MaxPre)→main runreaction window size→main run V_(Max))

Since there is no certainty that the criterion of linearity will alsoprovide the best results for precision measurements and for controlrecovery, the relationship between the main run reaction window andV_(MaxPre) is now finely adjusted. The reference curve produced above isnot changed in this case, in order to avoid varying too many parametersat the same time.

Inter-assay and intra-assay precision measurements (intra-assay=inseries, inter-assay=on different days) are carried out for variousconcentration levels of samples, standards and controls, the main runV_(Max) determinations being carried out with reaction windows ofdifferent size. The outcome of this is that a main run reaction windowsize which gives the best precision is obtained for each measurement andthus for each V_(MaxPre). Combination of all the measurements results ina similar dependency as that in the case of linearity. FIG. 2 shows anexample of a profile.

The procedure, which is in principle the same, for measuring the controlmaterial gives the best correctness (FIG. 2).

It is now possible to select an assignment of main run reaction windowsize t_(Lin) to V_(MaxPre) which ensures not only good precision butalso good recovery of control nominal values. Table 1 shows the resultfor Ferritin.

Once this relationship between t_(Lin) and V_(MaxPre) has been defined,the calculation of the reference curve must be repeated with the newcriteria. The new method can now be characterized completely, as isshown in summary form in Table 1 using the example of Ferritin.

Table 1

The V_(MaxPre) method using the example of Ferritin

V_(MaxLin) Parameters for Ferritin

Minimum reaction window: 40 seconds

Initial run reaction window: 80 seconds

V_(MaxPre) t_(Lin) >25 bit/s → 80 seconds 20-25 bit/s → 240-160 seconds10-20 bit/s → 360-240 seconds <10 bit/s → 360 seconds

a) Low-concentration Sample with Minimum (FIG. 3)

Minimum found at: 38 seconds

V_(MaxPre): 0.270 bit/s (38-118 seconds)

t_(Lin): 360 seconds

V_(MaxLin): 0.123 bit/s (38-360 seconds)

b) Normal-concentration Samples without a Minimum (FIG. 3)

Minimum found at: 0 seconds

V_(MaxPre): 24.02 bit/s (0-80 seconds) t_(Lin): 175 seconds V_(MaxLin):23.64 bit/s (23-198 seconds)

It must be stressed that the parameters determined in this way arehighly specific for the test system. A test system includes theanalyzer, the measurement instructions and the reagents for determininga specific analyte, if necessary with batch-specific differences, and,of course, the sample material.

It is also possible to assess the linearity of the measurement curve foreach measurement using mathematical linearity criteria and to evaluateonly that part of the curve which corresponds to these predeterminedcriteria. However, this gives poor precision even with low analyteconcentrations.

A further option for determining V_(Max) would be to match a polynomialto the measurement curve of a given sample and then to form the firstderivative of the polynomial. The maximum of the first derivativeindicates the maximum rate of reaction, although it has been found that,particularly in reactions with a high level of scatter, a V_(Max)determined in this way cannot be relied on, as can be seen, for example,from Table 2 (polynomial).

TABLE 2 Determination of V_(Max) directly from the 3rd order polynomialConcen- tration Polynomial Integral Dilution 1: [μg/1] MW [bit/s] VK [%]MW [bit/s] VK [%] 2.5 137.60 25.50 2.0 25.24 2.0 5 68.80 18.09 3.4 17.653.4 10 34.40 9.61 4.8 9.44 4.9 20 17.20 4.91 3.3 4.61 3.0 40 8.60 2.457.8 2.11 2.2 80 4.30 1.19 7.6 0.98 4.8 160 2.15 0.58 16.3 0.46 3.5 3201.08 0.34 38.6 0.22 9.7 640 0.54 0.17 48.6 0.09 15.0

Surprisingly, it is has been possible to show that, by adaptation of atest-specific integral area below the first derivative of thepolynomial, a measurement time period t_(Lin) can be determined in whichthe measurement curve can be evaluated using linear regression and leadsto a result which, with regard to linearity, precision and correctness,is equivalent to the method described above. The size of the integralarea is test-specific, i.e. it is preferably determined empirically foreach test method and each analyte, and possibly for specific testequipment as well.

For instance, it has been found to be possible to obtain optimum resultswith an integral area of 100 for various latex-enhanced tests on aspecific equipment (BN II, Behringwerke AG, Marburg), for example forFerritin and prostate-specific antigen (FIGS. 4a and b). With the sameequipment, the best integral area obtained for tests that are notlatex-enhanced (FIGS. 4d and e) is 10. With a different equipment (BCS,Behringwerke AG, Marburg), the best area obtained for the latex-enhancedtest with a photometric measurement is 20 (FIG. 4c).

The empirical determination of the optimum integral area is carried outin a seperate run as the determination of the dependency betweenV_(MaxPre) and t_(Lin), but the integral area determined for thisvariant is a constant which applies to all concentrations. The examplesin FIG. 4 each show a very high and a very low concentration. Thus, forexample, precision measurements were carried out in series for eachreference point to determine the precision of the various latex-enhancedtests using the Behring nephelometer (Behringwerke AG, Marburg).Linearity was obtained for each measurement by the correlationcoefficient of the linear regression. Correctness was determined bymeasuring control serums and convolution on a reference curve, likewiseevaluated using the integral method.

The integral area determined in this way is fitted underneath the firstderivative of each determination such that the first derivative of thepolynomial is the boundary of the area at the top and, at the bottom,the intersection of the lower boundary straight lines with the curve onthe X-axis indicates where the start and the end of the measurement timeperiod are located (FIG. 5).

The polynomial is calculated from the pairs of reaction kinetics valuesusing the least-squares principle. The system of normalized equations issolved using the Gaussian algorithm, a method which is known per se tothe person skilled in the art for solving non-linear regressions. Thematched polynomial is advantageously 3rd order, but higher orderpolynomials can also be used.

The following examples explain the invention:

COMPARATIVE EXAMPLE

a) Peak Rate Method

The Behringwerke TurbiTime System is a good example of a kineticevaluation method (DE 33 47 162). It has a measurement chamber in whichthe reaction in each case takes place in a single cell. The measurementsof the optical density continue until the maximum rate of reactionV_(Max), the result of the measurement, is reached, and are thenterminated.

The reagents for the system are set such that they allow a rapidreaction and, in the ideal case, the measurement can be terminated afterjust a few seconds. The time when V_(Max) is reached is also evaluated.This allows a decision to be made on the side of the Heidelberger curvethat is involved, and an incorrectly excessively low result can bevirtually precluded.

b) Fixed Time Method

The Behringwerke nephelometers (BN, BN100, BNII) measure the increase inturbidity as light scatter. The evaluation method is called “fixed time”on the basis of the fixed reaction times. The result of the measurementis the turbidity difference between an initial value and a final value.The nephelometers are fully automatic systems in which the samplesincubate simultaneously in a large number of cells, in order to allow ahigh throughput. The cells are moved past the measurement optics atregular intervals by a rotor in order to record the measurements.Reaction kinetics are thus obtained with measurement points atrelatively long intervals, for example 16 seconds in the case of the BNII.

EXAMPLE 1 The New V_(MaxLin) Method with 2-stage Evaluation

The method has two stages (FIG. 3, Tab. 1):

1. Determination of the optimum length of the main run reaction window(t_(Lin))

In a first run, the maximum rate of reaction V_(MaxPre) is sought overthe profile of the entire kinetics using a small initial run reactionwindow. The size of the window is defined specifically for therespective test such that it is still within the linear region of thereaction even with high antigen concentrations. The rate of reactiondetermined in this way is still relatively inaccurate.

It is used to define the ideal size of the main run reaction window(t_(Lin)) for this antigen concentration. In this context, ideal meansthat virtually the entire part of the reaction which has a linearprofile is used to define the rate of reaction. The reference table forthe assignment of the rate of reaction and size of the main run reactionwindow is likewise determined empirically on a test-specific basis.(FIG. 2, Tab. 1).

2. Determination of the maximum rate of reaction (V_(MaxLin))

a) Identification of the Minimum

At the start of a kinetic process, it is possible for the turbidity tofall before the actual reaction starts. This would result in anincorrectly excessively low V_(MaxPre) being determined for a longt_(Lin) (for example 360 seconds). This prevents the minimum beingidentified.

A very short reaction window is used to define the starting time of thereaction. In the example in FIG. 3 and Table 1, this is a time period of40 seconds. The reaction window starts from time zero and continues aslong as the gradient is negative. As soon as it reaches the firstpositive value, the first measurement point of the reaction window isalso the first value which can be considered for the calculation ofV_(MaxLin).

b) Search for V_(MaxLin)

The main run reaction window (t_(Lin)) determined in the initial run isused to look for the maximum rate of reaction over the entire profile.If (t_(Lin)) is greater than the actual reaction duration, the rate isaveraged over the entire reaction (from the minimum to the end of thereaction time).

The rate of reaction V_(MaxPre) determined in this way is used toproduce the reference curves, and the reference curve is used tocalculate the concentrations.

EXAMPLE 2 V_(MaxLin) Method with Integral Evaluation for the FerritinTest

The example uses the same pairs of measurements for Ferritin as Example1.

a) Identification of the Minimum

The identification of the minimum is carried out as is described forExample 1 in 2a). For the low-concentration sample (Table 1), that is tosay, a minimum is once again found at 38 seconds, and for thehigh-concentration sample at 0 seconds. All the measurement pointsbefore the minimum are now no longer considered.

b) Polynomial Matching

A third order polynomial is matched to the curve after the minimum, andits first derivative is formed (see also FIG. 5). The maximum of thefirst derivative occurs at 153 seconds for the low-concentration sample,and at 87 seconds for the high-concentration sample.

c) Finding the Evaluation Region t_(Lin)

An integral area underneath the maximum of the first derivative isenlarged iteratively until the limit, in this example 100, is reached(see also FIG. 5). At the same time, the area to the left and right ofthe time of the maximum of the first derivative is enlarged separatelyuntil the nominal area is reached. The reaction time window is obtainedin this way. If the nominal area on the right-hand side does not extendto the last measurement point, the right-hand evaluation region extendsto the last measurement point. If the nominal area on the left-hand sidedoes not extend as far as the minimum, the reaction time window thereextends to the minimum.

For the low concentration, the reaction time window is the same as forthe V_(MaxPre) method with 2 stages of evaluation from 38 to 360seconds, and for the high concentration the reaction time window extendsfrom 0 to 196 seconds.

d) Determination of the Raw Value

The raw value is obtained by linear regression within the reaction timewindow. This is 0.123 bits/second for the low concentration and 23.46bits/second for the high concentration, in comparison with 23.64bits/second in Example 1.

This value is either entered on a reference curve or is used with anexisting reference curve to determine a concentration.

The elementary advantage of the V_(MaxLin) method over the prior art isthe ideal matching of the reaction window to the respective reactionkinetics by the two-stage method or the integral method, which usesempirically defined, test-specific parameters. If necessary, they couldeven be defined on a batch-specific basis.

The very specific matching ensures that a maximum number of measurementpoints are used in each case for evaluation. This advantage isparticularly evident when the signal-to-noise ratio is large, as is thecase for the latex-enhanced tests on the BNII and, particularly in thatcase, with low analyte concentrations. The signal precision is betterthan that with the fixed time method, which uses only the differencebetween the initial and final values (FIG. 6).

The peak rate method is matched to kinetics, whose profiles haveseparations between a lag phase, the maximum rate of reaction and thesaturation region (U.S. Pat. No. 4,157,871). In the case of the reactionprofiles for the latex tests on the BNII (for example N Latex Ferritin),the lag phase and the saturation phase can often scarcely be identified(FIGS. 1a-1 c). At the start of the reaction, the first measurement istaken too late and the frequency at which the measurements are taken isalso too low for the lag phase still to be recorded. After 6 minutes,the saturation region is not reached by a long way.

If the three phases are not clearly separated and,in addition, thefrequency at which the measurements are taken is relatively low, thereis a risk that peak rate methods will incorrectly give excessively highmaximum rates. Particularly withlow-concentration samples, such as thatfor example in FIGS. 1a-1 c, there is clearly no point in determining apeak rate, since the rate of reaction is constant at a low level. Thisis also shown by the comparison of the accuracies with a short timewindow of 80 seconds, with which peak rates are still detected, and areaction window of 360 seconds (FIG. 7).

A range of criteria have been used to assess the new evaluation methodcomparatively (Table 3). In all the experiments, the same kinetics wereevaluated using the conventional fixed time method and the new method.In this case, the test time was shortened to 6 minutes from the previous12 minutes for V_(MaxLin), and the reference curve was extended. Thisresulted in a measurement range from 2.5 to 700 μg/l instead of 5-350μg/l (initial sample dilution 1:5, FIG. 11).

Table 3

Ferritin—test characteristics in comparison with the fixed time andV_(MaxLin) evaluation methods

The LogitLog function was used for the reference curve for both methods.Each result represents the mean value of 10 measurements. Furtherdescriptions can be found in the text.

TABLE 3 Fixed Time V_(MaxLin) Test time 12 Minutes 6 Minutes Lowermeasurement range limit 5 μg/l 2.5 μg/l Upper measurement range limit350 μg/l 700 μg/l Antigen excess certainty up to 25000 μg/l 50000 μg/lDilution trueness of samples Recovery 1:20 to 1:5 300-340 μg/l +16.0%+6.6% 553-665 μg/l —* +6.7% Intra Assay Precision Standard 1:2.5 1.8%1.8% 1:5 2.8% 3.6% 1:10 4.9% 5.0% 1:20 2.8% 2.7% 1:40 2.4% 2.1% 1:804.0% 4.5% 1:160 4.8% 3.4% 1:320 14.8% 9.6% 1:640 31.9% 15.3% SamplesMean value (10) 4.7% 3.4% Controls High (153 μg/l) 2.9% 3.2% Low (20.2μg/l 6.1% 4.6% Inter Assay Precision Samples Mean value (5) 2.0% 1.8%Controls High (153 μg/l) 4.0% 2.6% Low (20.2 μg/l) 4.6% 2.9% Controlrecovery High (153 μg/l) +2.2% +3.6% Low (20.2 μg/l) +2.4% +3.8%*outside the measurement range

Correlation (50 samples): V_(MaxLin)=0 951* Fixed Time—0.2785

The precision is equally good both in the series (intra assay) andbetween days (inter assay) for high and medium analyte concentrations.For low analyte concentrations, the precision for V_(MaxPre) isconsiderably better than for the conventional method (Table 3).

The dilution trueness is considerably better for V_(MaxLin) (Tab. 3,FIG. 9). Particularly in the case of samples whose concentration is atthe upper end of the measurement range, a repeat measurement at the nexthigher sample dilution (1:20) leads to values which are, on average,higher by 16%. The same experiment with the concentration twice as highfor the extended measurement range leads to only 7% higherconcentrations with V_(MaxLin). FIG. 9 also shows the improved dilutiontrueness.

The control recovery is similarly good for both evaluation methods(Table 3). In this case, it must be remembered that the nominal valuedetermination was carried out using the fixed time method, so thatconversion here allows still better recovery to be expected.

The correlation of the results from both methods is very good (FIG. 10).

The antigen excess certainty is ensured up to about 25,000 g/l for fixedtime and up to about 50,000 g/l for V_(MaxPre) (FIG. 11).

The test on PSA (prostate-specific antigen) can be used as a furtherexample. As can be seen from FIG. 8, equally good reference curves canbe produced using the V_(MaxLin) method and promise that the measurementrange can be extended upwards. In this case, the measurement time forV_(MaxPre) is only 6 minutes, in comparison with 18 minutes for thefixed time method.

Table 4 compares the intra-assay precision (CV, %) from 10 measurementsof the standard with fixed time, integral and two-stage evaluation forvarious latex-enhanced tests.

The two variants of the V_(MaxLin) method are roughly equivalent andgive considerably better accuracies than the fixed time method for lowconcentrations.

Table 4

A nominal area of 100 was used for the integral method

n.d.—value was not determined

*—mean value without zero standard

Measurement Time:

FRT 6 minutes(Integral and 2 stage)and 12 minutes (Fixed-time)

PSA 12 minutes (Integral and 2 stage) and 18 minutes (Fixed-time)

BR 9 minutes (Integral and 2 stage) and 12 minutes (Fixed-time)

RF 6 minutes (Integral and 2 stage) and 6 minutes (Fixed-time)

Dilution Integral 2 stage Fixed-time 1: FRT PSA BR RF FRT PSA BR RF FRTPSA BR RF  2.5 2.0 1.7 1.8 2.1 2.2  5 3.4 4.2 3.6 3.0 2.8 2.5  10 4.92.5 2.6 5.5 5.0 2.2 n.d. 4.1 4.9 2.4 3.7  20 3.0 4.5 2.9 3.1 2.7 5.4n.d. 2.9 2.8 3.1 2.2 3.2  40 2.2 1.7 4.3 5.5 2.1 2.5 n.d. 5.0 2.4 1.14.1 5.3  80 4.8 2.7 6.3 6.4 4.5 2.8 n.d. 6.1 4.0 2.2 5.4 6.4 160 3.5 1.94.0 5.7 3.4 2.8 n.d. 4.2 4.8 1.8 4.9 8.5 320 9.7 2.3 8.2 9.6 3.8 n.d.14.8  2.1 5.5 640 15.0  1.7 7.8 15.3  3.9 n.d. 2.0 13.6  Zero standard16.9  17.9  32.1  Mean value 5.4 2.5 5.2 4.6 5.3 3.3 n.d. 3.9 5.2 2.15.4 4.5

EXAMPLE 3

(FIG. 12)

V_(MaxLin) Method with Integral Evaluation for an aPTT Test

The evaluation is carried out for the Behring original test such thatthe time is determined at which a specific light absorption threshold isexceeded (fixed absorbance evaluation). It has been possible to showthat the V_(MaxPre) method with integral evaluation can be used just aswell. The signal difference between the standard and the 1:3 dilutedstandard (125.7 mE/second compared with 28.2 mE/second) is higher thanfor the fixed absorbance evaluation (33.9 seconds compared with 81.4seconds). It is advantageous that no threshold need be exceeded for theV_(MaxPre) evaluation (system: BCS, Behring reagent OQGS, Behring testnumber: 28, nominal area: 10, polynomial order: 5, evaluation range:30-100 seconds (undiluted standard), 70-150 seconds (1:3 standard), FIG.12).

For V_(MaxPre) evaluation of tests with a long lag phase, the startingtime for the evaluation must be kept variable. This is done, forexample, by an algorithm looking for a positive limit gradient in ananalogous manner to the search for a minimum, the value of whichgradient must be definable and depends on the test system. When thelimit gradient is reached, the start of the search time window providesthe starting time for the V_(MaxLin) evaluation.

EXAMPLE 4

(FIG. 12)

V_(MaxLin) Method with Integral Evaluation for an ATIII Test

The ATIII test, in which a chromogenic substrate is converted by thethrombin that is not inhibited by ATIII, can also be evaluated usingV_(MaxLin) (System: BCS, Behring reagent OWWR, Behring test number: 28,evaluation range: 0-45 seconds, nominal area: 1, polynomial order: 5).When measurements were carried out ten times, the accuracies wereroughly just as good as those from conventional evaluation (slope from15-45 seconds).

EXAMPLE 5

(FIG. 12)

V_(MaxLin) Method with Integral Evaluation for a Platelet AggregationTest

Widely different reaction kinetics may occur for the aggregation ofblood platelets. The light transmission of the plasma increases in thecourse of aggregation. Using the example in FIG. 12, the profiles showthe cell aggregation of a sample 2 hours and 10 hours after takingblood. In both profiles, V_(MaxLin) with integral evaluation finds thelinear section very well and determines an aggregation rate of 22.4 forthe fresh sample and 14.5 for the old sample (System: BCT, Sample: 50 μlADP (1.25 μM) from Sigma, St. Louis, USA, mixed with 150 μl ofplatelet-rich plasma, evaluation range: 7-80 seconds, nominal area: 5,polynomial order: 5).

EXAMPLE 6

(FIG. 13)

V_(MaxLin) Method with Integral Evaluation for the Von-Willebrand Test

The Behring Von-Willebrand test determines the Von-Willebrandfactor-dependent platelet aggregation using a reagent which containsfixed platelets and Ristocetin. The evaluation is carried out with theBehring original test such that the time is determined at which aspecific threshold (100 mE) of the light absorption is undershot (fixedabsorbance evaluation). The reference curve points may be determinedonly down to 20% Ristocetin co-factor concentration (standard humanplasma dilution) using the conventional fixed absorbance evaluationsince, below this, the threshold is no longer undershot because of theweak reaction, and thus it is no longer possible to determine any resultat all. In contrast, V_(MaxPre) with integral evaluation permits areference curve down to about 5% for the same measurements (System: BCT,Behring reagent OUBD, Behring test number 390, evaluation range 7-80seconds, nominal area: 5, polynomial order: 5).

What is claimed is:
 1. A method for an instrument determination of themaximum rate value, V_(Maxlin), of a measured variable, L(t), whichchanges with time, t, in a region with a linear profile using time andthe period of a time window as variables within said region with saidlinear profile, comprising: (a) measuring, in at least one preliminaryexperiment, said measured variable L(t); (b) determining a relationshipbetween the maximum rate value of said measured variable L(t) from saidat least one preliminary experiment, V_(MaxPre), and the period of theoptimum time window, t_(Lin); (c) measuring, in at least one main runexperiment, said measured variable L(t); (d) determining the value ofV_(MaxPre) for said at least one main run experiment; (e) determiningthe value of the period of the optimum time window t_(Lin) by inputtingthe value of V_(MaxPre) of step (d) into said relationship of step (b)for said at least one main run experiment; and (f) determiningV_(MaxLin), the maximum rate value of said L(t) measured in step (c)within said optimum time window of step (e).
 2. A method according toclaim 1, wherein, in steps (a) and (c), reacting a sample with at leastone reagent leads to a time-dependent change of said measured variableL(t).
 3. A method according to claim 2, wherein said sample comprises atleast one analyte.
 4. A method according to claim 3, wherein theconcentration of said at least one analyte correlates to the maximumrate of said measured variable L(t).
 5. A method according to claim 3,wherein said at least one analyte and said at least one reagent arepartners in a specific bonding pair.
 6. A method according to claim 5,wherein said at least one analyte and said at least one reagent areindividually chosen from antigens and antibodies.
 7. A method accordingto claim 3, wherein said at least one analyte is chosen from plasmaproteins.
 8. A method according to claim 3, wherein said at least oneanalyte is a haptene.
 9. A method according to claim 1, wherein saidL(t) is only partially linear.
 10. A method according to claim 1,wherein determining said relationship in step (b) utilizes a previouslydetermined, test-specific reaction time window t_(Test).
 11. A methodaccording to claim 1, wherein determining the value of V_(MaxPre) instep (d) utilizes a previously determined, test-specific reaction timewindow t_(Test).
 12. A method according to claim 1, further comprisingperforming a mathematical fit to said measured variable L(t).
 13. Amethod according to claim 12, further comprising performing a firstderivative of said mathematical fit.
 14. A method according to claim 13,wherein determining said relationship in said step (b) utilizes saidfirst derivative.
 15. A method according to claim 13, whereindetermining the value of V_(MaxPre) in said step (d) utilizes said firstderivative.
 16. A method according to claim 12, wherein saidmathematical fit is a polynomial fit.
 17. A method according to claim 1,wherein said measured variable is the turbidity.
 18. A method accordingto claim 1, wherein said measured variable is light scatter.
 19. Amethod according to claim 1, wherein said method is for coagulationanalysis.
 20. A method for an instrument determination of the maximumrate value, V_(MaxLin), of a measured variable, L(t), which changes withtime, t, in a region with a linear profile using time and the period ofa time window as variables within said region with said linear profile,comprising: (a) measuring, in at least one separate run experiment, saidmeasured variable L(t); (b) determining the nominal area correspondingto the period of the optimum time window under the maximum value of thefirst derivative of said measured variable L(t); (c) measuring, in atleast one main run experiment, said measured variable L(t); (d)determining the time of the maximum value of the first derivative ofsaid measured variable L(t) of said at least one main run experiment;(e) determining the value of the period of the optimum time windowt_(Lin) by expanding the nominal area under the maximum value of thefirst derivative of step (d) until the value of said nominal area ofstep (b) is reached to set the time period on both sides of the time ofstep (d); and (f) determining V_(MaxLin), the maximum of said L(t)measured in step (c) within said optimum time window of step (e).
 21. Amethod according to claim 20, wherein, in steps (a) and (c), reacting asample with at least one reagent leads to a time-dependent change ofsaid measured variable L(t).
 22. A method according to claim 21, whereinsaid sample comprises at least one analyte.
 23. A method according toclaim 22, wherein the concentration of said at least one analytecorrelates to the maximum rate of said measured variable L(t).
 24. Amethod according to claim 22, wherein said at least one analyte and saidat least one reagent are partners in a specific bonding pair.
 25. Amethod according to claim 24, wherein said at least one analyte and saidat least one reagent are individually chosen from antigens andantibodies.
 26. A method according to claim 22, wherein said at leastone analyte is chosen from plasma proteins.
 27. A method according toclaim 22, wherein said at least one analyte is a haptene.
 28. A methodaccording to claim 20, wherein said L(t) is only partially linear.
 29. Amethod according to claim 20, further comprising performing amathematical fit to said measured variable L(t).
 30. A method accordingto claim 29, further comprising performing a first derivative of saidmathematical fit.
 31. A method according to claim 29, wherein saidmathematical fit is a polynomial fit.
 32. A method according to claim20, wherein said measured variable is the turbidity.
 33. A methodaccording to claim 20, wherein said measured variable is light scatter.34. A method according to claim 20, wherein said method is forcoagulation analysis.
 35. A method according to claim 20, furthercomprising performing a mathematical fit to said measured variable L(t).36. A method according to claim 35, further comprising performing afirst derivative of said mathematical fit.
 37. A method according toclaim 37, wherein determining said nominal area in said step (b)utilizes said first derivative.
 38. A method for an instrumentdetermination of the maximum rate value, V_(MaxLin), of a measuredvariable, L(t), which changes with time, t, in a region with a linearprofile using time and the period of a time window as variables withinsaid region with said linear profile, comprising: (a) measuring, in atleast one separate run experiment, said measured variable L(t); (b)measuring, in at least one main run experiment, said measured variableL(t); (c) at least one step for determining the period of the optimumtime window t_(Lin); and (d) determining V_(MaxLin), the maximum of saidL(t) measured in step (b) within said optimum time window of step (c).